Glucocorticoid (GC)-induced leucine zipper (GILZ) has been shown to mediate or mimic several actions of GC. This study assessed the role of GILZ in self-resolving and GC-induced resolution of neutrophilic inflammation induced by LPS in mice. GILZ expression was increased during the resolution phase of LPS-induced pleurisy, especially in macrophages with resolving phenotypes. Pretreating LPS-injected mice with trans-activator of transcription peptide (TAT)–GILZ, a cell-permeable GILZ fusion protein, shortened resolution intervals and improved resolution indices. Therapeutic administration of TAT-GILZ induced inflammation resolution, decreased cytokine levels, and promoted caspase-dependent neutrophil apoptosis. TAT-GILZ also modulated the activation of the survival-controlling proteins ERK1/2, NF-κB and Mcl-1. GILZ deficiency was associated with an early increase of annexin A1 (AnxA1) and did not modify the course of neutrophil influx induced by LPS. Dexamethasone treatment resolved inflammation and induced GILZ expression that was dependent on AnxA1. Dexamethasone-induced resolution was not altered in GILZ−/− mice due to compensatory expression and action of AnxA1. Our results show that therapeutic administration of GILZ efficiently induces a proapoptotic program that promotes resolution of neutrophilic inflammation induced by LPS. Alternatively, a lack of endogenous GILZ during the resolution of inflammation is compensated by AnxA1 overexpression.

Objective. Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid-induced protein, the reported molecular interactions of which suggest that it functions to inhibit inflammation. However, the role of endogenous GILZ in the regulation of inflammation in vivo has not been established. This study was undertaken to examine the expression and function of GILZ in vivo in collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis (RA), and in RA synoviocytes.Methods. GILZ expression was detected in mouse and human synovium by immunohistochemistry and in cultured cells by real-time polymerase chain reaction and permeabilization flow cytometry. GILZ function was assessed in vivo by small interfering RNA (siRNA) silencing using cationic liposome-encapsulated GILZ or control nontargeting siRNA and was assessed in vitro using transient overexpression.Results. GILZ was readily detectable in the synovium of mice with CIA and was up-regulated by therapeutic doses of glucocorticoids. Depleting GILZ expression in vivo increased the clinical and histologic severity of CIA and increased synovial expression of tumor necrosis factor and interleukin-1 (IL-1), without affecting the levels of circulating cytokines or anticollagen antibodies. GILZ was highly expressed in the synovium of patients with active RA and in cultured RA synovial fibroblasts, and GILZ overexpression in synovial fibroblasts inhibited IL-6 and IL-8 release.Conclusion. Our findings indicate that GILZ functions as an endogenous inhibitor of chronic inflammation via effects on cytokine expression and suggest that local modulation of GILZ expression could be a beneficial therapeutic strategy.Chronic inflammation in rheumatoid arthritis (RA) is mediated by complex interactions of macrophages, T cells, and resident cells through the effects of multiple proinflammatory cytokines. Proinflammatory events are balanced by antiinflammatory regulatory pathways, one of the most important of which is mediated by endogenous glucocorticoids. During inflammation, circulating cytokines stimulate the hypothalamicpituitary-adrenal axis, resulting in the release of endogenous glucocorticoids and subsequent inhibition

Glucocorticoids have been exploited therapeutically for more than six decades through the use of synthetic glucocorticoids as anti-inflammatory agents, and are still used in as many as 50% of patients suffering from inflammatory diseases such as rheumatoid arthritis (RA). Better understanding of the mechanisms of action of glucocorticoids could enable the development of therapies that dissociate the broad-spectrum benefits of glucocorticoids from their adverse metabolic effects. The glucocorticoid-induced leucine zipper protein (GILZ; also known as TSC22 domain family protein 3) is a glucocorticoid-responsive molecule whose interactions with signal transduction pathways, many of which are operative in RA and other inflammatory diseases, suggest that it is a key endogenous regulator of the immune response. The overlap between the observed effects of GILZ on the immune system and those of glucocorticoids strongly suggest GILZ as a critical mediator of the therapeutic effects of glucocorticoids. Observations of the immunomodulatory effects of GILZ in human RA synovial cells, and in an in vivo model of RA, support the hypothesis that GILZ is a key glucocorticoid-induced regulator of inflammation in RA. Moreover, evidence that the effect of GILZ on bone loss might be in contrast to those of glucocorticoids suggests manipulation of GILZ as a potential means of dissociating the beneficial anti-inflammatory effects of glucocorticoids from their negative metabolic repercussions.

Objective. Glucocorticoid-induced leucine zipper (GILZ) has effects on inflammatory pathways that suggest it to be a key inhibitory regulator of the immune system, and its expression is exquisitely sensitive to induction by glucocorticoids. We undertook this study to test our hypothesis that GILZ deficiency would exacerbate experimental immune-mediated inflammation and impair the effects of glucocorticoids on inflammation and, correspondingly, that exogenous GILZ would inhibit these events.Methods. GILZ ؊/؊ mice were generated using the Cre/loxP system, and responses were studied in delayedtype hypersensitivity (DTH), antigen-induced arthritis (AIA), K/BxN serum-transfer arthritis, and lipopolysaccharide (LPS)-induced cytokinemia. Therapeutic expression of GILZ via administration of recombinant adeno-associated virus expressing the GILZ gene (GILZ-rAAV) was compared to the effects of glucocorticoid in collagen-induced arthritis (CIA).Results. Increased T cell proliferation and DTH were observed in GILZ ؊/؊ mice, but neither AIA nor K/BxN serum-transfer arthritis was affected, and GILZ deficiency did not affect LPS-induced cytokinemia. Deletion of GILZ did not impair the effects of exogenous glucocorticoids on CIA or cytokinemia. In contrast, overexpression of GILZ in joints significantly inhibited CIA, with an effect similar to that of dexamethasone. Conclusion. Despite effects on T cell activation, GILZ deficiency had no effect on effector pathways of arthritis and was unexpectedly redundant with effects of glucocorticoids. These findings do not support the hypothesis that GILZ is central to the actions of glucocorticoids, but the efficacy of exogenous GILZ in CIA suggests that further evaluation of GILZ in inflammatory disease is required.

Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory protein first identified in T lymphocytes. We recently observed that GILZ is highly expressed in synovial endothelial cells in rheumatoid arthritis. However, the function of GILZ in endothelial cells is unknown. To investigate the actions of GILZ in this cell type, we induced GILZ expression in HUVECs via transient transfection. GILZ overexpression significantly reduced the capacity of TNF-stimulated HUVECs to support leukocyte rolling, adhesion, and transmigration. These effects were associated with decreased expression of E-selectin, ICAM-1, CCL2, CXCL8, and IL-6. Experiments in a human microvascular endothelial cell line demonstrated that TNF-inducible NF-κB activity was significantly inhibited by overexpression of GILZ. Exogenous GILZ inhibited TNF-induced NF-κB p65 DNA binding, although this occurred in the absence of an effect on p65 nuclear translocation, indicating that the mechanism of action of exogenous GILZ in endothelial cells differs from that reported in other cell types. GILZ overexpression also inhibited TNF-induced activation of p38, ERK, and JNK MAPKs, as well as increased expression of the MAPK inhibitory phosphatase, MKP-1. In contrast, silencing endogenous GILZ in glucocorticoid-treated HUVECs did not alter their capacity to support leukocyte interactions. These data demonstrate that exogenous GILZ exerts inhibitory effects on endothelial cell adhesive function via a novel pathway involving modulation of NF-κB p65 DNA binding and MAPK activity. Induction of GILZ expression in endothelial cells may represent a novel therapeutic modality with the potential to inhibit inflammatory leukocyte recruitment.

Immunoblotting sera from 26 patients with septicemia due to an epidemic strain of methicillin-resistant Staphylococcus aureus (EMRSA-15), 6 of whom died, revealed an immunodominant EMRSA-15 antigen at 61 kDa. There was a statistically significant correlate (P < 0.001) between survival and immunoglobulin G to the 61-kDa band. The antigen was identified by sequencing positive clones obtained by screening a genomic expression library of EMRSA-15 with pooled sera from patients taken after the septicemic episode. Eluted antibody reacted with the 61-kDa antigen on immunoblots. The amino terminus was obtained by searching the S. aureus NCTC 8325 and MRSA strain COL databases, and the whole protein was expressed in Escherichia coli TOP 10F. The derived amino acid sequence showed homology with ABC transporters, with paired Walker A and Walker B motifs and 73% homology to YkpA from Bacillus subtilis. Epitope mapping of the derived amino acid sequence with sera from patients who had recovered from EMRSA-15 septicemia delineated seven epitopes. Three of these epitopes, represented by peptides 1 (KIKVYVGNYDFWYQS), 2 (TVIVVSHDRHFLY NNV), and 3 (TETFLRGFLGRMLFS), were synthesized and used to isolate human recombinant antibodies from a phage antibody display library. Recombinant antibodies against peptides 1 and 2 gave logarithmic reductions in organ colony counts, compared with control groups, in a mouse model of the infection. This study suggests the potential role of an ABC transporter as a target for immunotherapy.

Objective Mesenchymal stem cells (MSCs) are potent immunosuppressive cells that have shown promise in the treatment of rheumatoid arthritis (RA). Deciphering the intrinsic characteristics of MSCs that correlate with their biologic activity will facilitate their clinical use. Recently, the role of glucocorticoid‐induced leucine zipper (GILZ) in the development of RA has been documented. The aim of this study was to evaluate whether GILZ expression by MSCs may contribute to their therapeutic effect. Methods MSCs were isolated from GILZ‐deficient (GILZ−/−) mice and wild‐type mice. MSCs (1 × 106 cells) were injected twice via the tail vein into mice with collagen‐induced arthritis (CIA). Results In vitro, we showed that GILZ is a key factor involved in the immunosuppressive potential of MSCs. MSCs derived from GILZ−/− mice did not suppress the proliferation of CD4+ T cells and were less efficient than MSCs derived from WT mice in altering Th17 cell polarization. Thus, we investigated the role of GILZ in an experimental model of arthritis and demonstrated that although WT MSCs significantly reduced paw swelling in arthritic mice, GILZ−/− MSCs did not. Moreover, the magnitude of the effects of GILZ−/− MSCs on Th17 cell frequency was significantly lower than that of WT MSCs. The therapeutic effect of MSCs correlated with the generation of Treg cells bearing the CD4 + RORγt+IL‐17low IL‐10+ signature, and Th17 cell polarization was GILZ dependent. Conclusion This study demonstrates that GILZ has an essential role in the therapeutic effectiveness of MSCs in arthritis by favoring Th17 cell polarization toward a regulatory phenotype. Therefore, potentiation of GILZ expression in MSCs could represent a means to enhance their therapeutic effect in autoimmune diseases.

Objectives Glucocorticoids remain a mainstay of the treatment of rheumatoid arthritis (RA). Dose-dependent adverse effects highlight the potential for therapies that regulate glucocorticoid sensitivity to enable glucocorticoid dose reduction. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory protein implicated in the pathogenesis of RA, which also impairs glucocorticoid sensitivity via inhibition of the MAP kinase phosphatase, MKP-1. The intracellular protein glucocorticoid-induced leucine zipper (GILZ) mimics the effects of glucocorticoids in models of RA, but whether it represents a target for the modulation of glucocorticoid sensitivity remains unknown. We therefore investigated whether GILZ is implicated in regulation of glucocorticoid sensitivity by MIF. Methods GILZ expression was studied in the presence and absence of MIF, and the role of GILZ in the MIF-dependent regulation of the glucocorticoid sensitivity mediator MAP kinase phosphatase-1 (MKP-1) studied at the level of expression and function. Results GILZ expression was significantly inhibited by endogenous MIF, both basally and during responses to glucocorticoid treatment. The effects of MIF on GILZ were dependent on the expression and Akt-induced nuclear translocation of the transcription factor FoxO3a. GILZ was shown to regulate the expression of MKP-1, and consequent MAP kinase phosphorylation and cytokine release. Conclusions MIF exerts its effects on MKP-1 expression and MAP kinase activity through inhibitory effects on GILZ. These findings suggest a previously unsuspected intersection between these two molecules and identify GILZ as a potential target for the therapeutic regulation of glucocorticoid sensitivity.